The present invention, in some embodiments thereof, relates to microorganisms which express enzymes which catalyze the conversion of formate and acetyl-CoA to pyruvate.
The concept of biorefineries has become a wide spread notion in the last decade. It relies on the premise that living organisms can and should be used to supply the increasing demand by humanity for specialized chemicals, including fuels, solvents, plastics, pharmaceuticals, etc. Today, most of these chemicals are derived, directly or indirectly, from fissile carbons. However, with the imminent depletion of these fossil carbons and the increase in atmospheric CO2 it has become essential to find alternative sources for these important materials.
The suggested feedstocks for most of the proposed biorefineries are simple sugars, starch, or lingocellulosic biomass. While the latter alternative has an apparent advantage over the former by not-competing with human consumption needs, it still presents numerous difficulties, including a problematic fermentation technology and feedstock availability and transportation. A fascinating alternative feedstock would be electric current. Electrons can be shuttled from an electrode to living cells, providing the necessary reducing equivalents and energy to support autotrophic growth and electrosynthesis of desired commodities. Since electricity is widely available, microbial electrosynthesis can be spatially and temporally decoupled from energy production and can take place at any convenient location and time.
Microbial electrosynthesis can be especially useful for the renewable energy market. One major drawback of most renewable energy sources, including solar, wind, hydro and nuclear, is that they are hard to store in a convenient way. Microbial electrosynthesis of fuels can thus serve to address this problem efficiently, converting electrical energy to kinetically stable chemical bonds.
CO2 can be directly reduced at the cathode (the electrons are derived from water splitting at the anode), providing organic compounds that can be used by living cells as a source of reducing equivalents, energy and even carbon. A diverse group of compounds can be produced in this manner. The production of simple alcohols, such as methanol, ethanol and propanol, hydrocarbons, such as methane and ethylene, or acids with more than one carbon, such as acetic acid and oxalic acid, has the advantage of supplying microbes with compounds relatively simple to metabolize and/or being rich in reducing equivalents. However, the electrocatalytic production of all of these compounds is generally inefficient (not specific to a single product and/or requiring high overpotential), requiring costly catalysts and/or supporting low current density. In contrast, there are two compounds that can be produced by direct reduction of CO2 at relatively high efficiency (although lower than that of molecular hydrogen) and an acceptable current density: carbon monoxide and formic acid. Since carbon monoxide is a toxic and flammable gas with low solubility, formic acid, being readily soluble and of low toxicity, is a preferred mediator of electrons. In fact, a formate-based economy was recently proposed as an alternative to the hydrogen-based economy or methanol-based economy concepts.
In most organisms, glycolysis is the main route by which the cell obtains its biomass building blocks, as well as available energy (in the form of ATP), from sugars [1]. There are several natural variants of the glycolytic pathway, differing in their ATP yield, metabolic flux and sensitivity to external conditions [2-4]. However, all these alternatives share one feature in common: they produce stoichiometric amounts of pyruvate molecules from hexoses, pentoses and trioses. On the other hand, the faith of pyruvate changes depending on the organism and conditions. Under aerobic conditions or when other electron acceptors are available, pyruvate is mostly decarboxylated and oxidized—via either pyruvate dehydrogenase or pyruvate oxidase—to form acetyl-CoA, which feeds the TCA cycle. Other pyruvate molecules are used as biomass building blocks, either directly (e.g., valine biosynthesis) or through anaplerotic reactions (e.g., aspartate biosynthesis). When terminal electron acceptors are absent, requiring a redox-balanced fermentation, pyruvate is usually converted to lactate or ethanol, in a process which provides two ATP molecules per fermented hexose.
Mixed acid fermentation is a metabolic strategy which enables the cells to increase their ATP production from 2 to 3 molecules per hexose (e.g., [5-9]). In this redox-balanced process the enzyme pyruvate formate-lyase (PFL) cleaves pyruvate to acetyl-CoA and formate. Half of the acetyl-CoA molecules are then reduced to ethanol, while the other half is secreted as acetate, providing an extra ATP. PFL operates in numerous organisms [10], prokaryotes [6,7,11-15] as well as eukaryotes [16-18]. The most studied PFL variant is from the γ-proteobacterium Escherichia coli (e.g., [19-22]).
In E. coli, PFL is encoded by the pflB gene and is active as a homodimer of 85 kDa polypeptides [23]. Pyruvate cleavage takes place via a radical mechanism, which involves a glycyl radical on G734 of PFL [24-26]. Pyruvate formate-lyase activating enzyme (PFL-AE)—encoded by the pflA gene in E. coli—generates the stable and catalytically essential glycyl radical [27,28]. PFL-AE performs this remarkable feat by using an iron-sulfur cluster and S-adenosylmethionine, thus placing it among the AdoMet radical superfamily of enzymes [28].
The glycyl radical is susceptible to destruction by oxygen, which results in irreversible cleavage of the polypeptide and inactivation of PFL [29,30]. However, previous studies have shown that E. coli cells grown under microaerobic conditions produce a significant amount of formate, indicating that PFL retains its activity in the presence of low level of oxygen [21,31,32]. The product of the yfiD gene was shown to reactivate PFL in the presence of oxygen by replacing its fragmented part [32,33].
While the PFL reaction was shown to be reversible in-vitro (ΔrG′o≈−10 kJ/mol at pH 7.5 and ionic strength of 0.2 M), catalyzing pyruvate formation at a non-negligible rate (kcat>4 s−1 [37]), the significance of this backward reaction was never explored in-vivo.
PCT International Application No. PCT/IL2013/050643 teaches formatotrophic bacteria that use the reductive glycine pathway.